Embryonic stem (ES) cell is a pluripotent cell derived from the inner cell mass (ICM) of a blastocyst. The ES cell can be unlimitedly cultured in an undifferentiated state, and differentiate into various cell types. According to studies regarding the in vitro differentiation of ES cell, the ES cell can be induced to differentiate into a nerve cell, a hematopoietic cell, and the endodermal cells of the pancreas, liver, and so on. The results of many studies suggest that, since the ES cell is able to recapitulate normal developmental processes, it can be used in the in vitro analysis of induction processes in developmental biology, the cell therapy, the application to hepatotoxicity, and the drug metabolism studies in drug discovery (Non-Patent Documents 1 and 2).
The liver is an important organ having complicated functions such as carbohydrate metabolism, urea and lipid metabolism, the storage of essential nutrition, and biotransformation of drugs. When the toxicity of a metabolite is stronger than that of a parent molecule, the biotransformation of drugs includes bioactivation as well as detoxication. Accordingly, the biotransformation of drugs plays an important role at the initial stage of a drug discovery process. A primary culture has a short lifespan, and thus the culture cannot be maintained for a long period of time. Moreover, the primary culture comprises a large extent of donor-derived mutation. The ES cell is an attractive material because large quantities of cells can always be prepared for the development of a strategy for screening for a novel drug.
The liver of a vertebrate animal develops from the ventral foregut endoderm. In addition to the liver, the lung, pancreas and thyroid also develop from the tissues of the ventral foregut endoderm (Non-Patent Documents 3 and 4). From the results of the previous studies, it was revealed that various sites of the ventral foregut are regionalized by signals from the mesoderm located adjacent to it. However, the mechanism whereby specific endodermal tissues develop has not yet been sufficiently clarified. According to the studies by Wells and Melton, signals from the mesectoderm regionalize the endoderm, and the endoderm then acquires a specific trait. Thereafter, signals derived from the notochord transmit strong signals to the dorsal pancreas. On the other hand, it has been found that FGF and BMP derived from the cardiac mesoderm and the septum transversum mesenchyme are essential for heart induction, the liver gene expression, and the growth of the endoderm (Non-Patent Documents 5 and 6). Cardiac mesoderm-derived FGF signals change the fate of the endoderm to the liver, but not to the pancreas (Non-Patent Document 7).
A study report has revealed that the ES cell had been induced to differentiate into a hepatic cell both in vitro and in vivo. The in vitro method includes the formation of an embryoid body that imitates a microenvironment necessary for the induction of the formation of a hepatic organ (Non-Patent Documents 8 and 9) and treatments with a specific growth factor or cytokine important for the differentiation into hepatocytes (Patent Document 10). Moreover, it has also been revealed that the ES cell differentiates into a hepatic cell by the co-culture of the ES cell and a fetal mesoderm derived cell (Patent Document 11). In recent studies, the in vitro production of stem cells derived from the ES cells using BMP4 has been reported (Non-Patent Document 12). Such study results are promising, but the differentiation into hepatocytes is insufficient. Accordingly, sufficient quantities and qualities of hepatocytes have not yet been produced, so fat
Furthermore, Patent Document 1 describes a method for inducing the differentiation of an ES cell into an endodermal cell, which comprises culturing a mammal-derived ES cell in the presence of a supporting cell.    Patent Document 1: International Publication WO2006/126574    Non-Patent Document 1: Davila, J. C., Cezar, G. G., Thiede, M., Strom, S., Mild, T. and Trosko, J. (2004). Use and application of stem cells in toxicology. Toxicol Sci 79, 214-23.    Non-Patent Document 2: Kulkarni, J. S. and Khanna, A. (2006). Functional hepatocyte-like cells derived from mouse embryonic stem cells: a novel in vitro hepatotoxicity model for drug screening. Toxicol In Vitro 20, 1014-22.    Non-Patent Document 3: Wells, J. M. and Melton, D. A. (1999). Vertebrate endoderm development. Annu Rev Cell Dev Biol 15, 393-410.    Non-Patent Document 4: Zaret, K. S. (2000). Liver specification and early morphogenesis. Mech Dev 92, 83-8.    Non-Patent Document 5: Jung, J., Zheng, M., Goldfarb, M. and Zaret, K. S. (1999). Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 284, 1998-2003.    Non-Patent Document 6 : Rossi, J. M., Dunn, N. R, Hogan, B. L. and Zaret, K. S. (2001). Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Dev 15, 1998-2009.    Non-Patent Document 7 : Deutsch, G., Jung, J., Zheng, M., Lora, J. and Zaret, K. S. (2001). A bipotential precursor population for pancreas and liver within the embryonic endoderm. Development 128, 871-81.    Non-Patent Document 8 : Asahina, K., Fujimori, H., Shimizu-Saito, K., Kumashiro, Y., Okamura, K., Tanaka, Y., Teramoto, K., Arii, S. and Teraoka, H. (2004). Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells. Genes Cells 9, 1297-308.    Non-Patent Document 9: Heo, J., Factor, V. M., Uren, T., Takahama, Y., Lee, J. S., Major, M., Feinstone, S. M. and Thorgeirsson, S. S. (2006). Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver. Hepatology 44, 1478-86.    Non-Patent Document 10: Teratani, T., Yamamoto, H., Aoyagi, K., Sasaki, H., Asari, A., Quinn, G., Sasaki, H., Terada, M. and Ochiya, T. (2005). Direct hepatic fate specification from mouse embryonic stem cells. Hepatology 41, 836-46.    Non-Patent Document 11: Ishii, T., Yasuchika, K., Fujii, H., Hoppo, T., Baba, S., Naito, M., Machimoto, T., Kamo, N., Suemori, H., Nakatsuji, N. et al. (2005). In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes. Exp Cell Res 309, 68-77.    Non-Patent Document 12 : Gouon-Evans, V., Boussemart, L., Gadue, P., Nierhoff, D., Koehler, C. I., Kubo, A., Shafritz, D. A. and Keller, G. (2006). BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol 24, 1402-11.